JPH01248062A - Immune detecting method - Google Patents

Abstract

PURPOSE:To shorten the detecting time, by increasing fluorescence which is extinguished by mixing a specific probe substance with an antibody, through introduction of a desired substance to be detected thereby to measure the same. CONSTITUTION:A probe substance B is mixed with a solution of an antibody A thereby to extinguish fluorescence of the antibody A. Then, an aiming substance C to be detected is added to the solution, so that the strength of the fluorescence is increased and detected. For the probe substance B, a substance (for example, a compound expressed by a formula II) in which a resembling substance (e.g. a compound represented by a formula I) coupling with the antibody A and an extinguishing substance (e.g. dinitrofluorobenzene) which functions to extinguish the fluorescence of the antibody A are chemically coupled is used. It is preferable that the substance C is methamphetamine, amphetamine or ephedrine. It is to be noted that a reference R1 in the formula I denotes H, hydroxyl group; R2 representing H, alkyl; X representing H, methoxy; and (n) representing a positive number not less than 0, 1, respectively.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to an immunological detection method that can be used mainly for the detection of pathogens or disease markers in clinical tests, and also for the wide range of industrial trace detection fields. .

Conventional technology Detection methods using naturally occurring or artificially produced antibodies are characterized by high specificity and high sensitivity;
It is currently used for the purpose of detecting target substances in extremely small amounts. Such purposes include, for example, clinical testing of pathogens in the blood, so-called disease markers that are secreted specifically during diseases such as tumors, myocardial infarction, and cerebral thrombosis, and detection of trace amounts of substances in the air. There are purposes for doing so. In recent years, for this purpose, for example, "Enzyme immunoassay method 3rd edition" by Eiji Ishikawa, Tadashi Kawai, and Kiyoshi Miyai (Igakushoin 19
Many types of immunoassay methods have been developed, as described in 1987, pp. 31-64). This method is divided into two types depending on whether the substance to be detected is a high molecule (protein) or a low molecule (hapten).

The present invention mainly targets hapten systems, and the experimental procedure of the ELISA method, which is a typical example of this method, will be explained step by step.

The antigen is coated with a carrier protein, such as serum albumin (BSA),
Funshugate, which is bound to a detection substance or a derivative with a functional group introduced thereto, is dissolved in a buffer to prepare an antigen solution.

Inject 100 μL/well of the antigen solution into a microplate (96-well plate made of vinyl chloride or polystyrene) and store at 20° C. overnight.

(To) Inject 250 μL/well of blocking BSA buffer solution,
Leave at room temperature for 0.5 to 2 hours. Then, wash with buffer or pure water 3 to 5 times.

(q Inject the antibody reaction detection substance solution and add more antibody solution while shaking. After storing at room temperature for 3 to 5 hours, remove the antibody solution with an aspirator and wash 3 to 5 times with buffer or pure water. do.

q A solution of an antibody against the antibody (second antibody) washed with a reaction enzyme of the second antibody, such as peroxidase, is injected and left at room temperature for 0.6 to 2 hours. Afterwards, the buffer solution is washed 3 to 5 times with pure water.

■ Substrate reaction and termination Dissolve a coloring agent, such as 0-phenylenediamine (for selenium detection) in a buffer, add a solution (substrate solution) containing 30% hydrogen peroxide immediately before use, and allow the coloring reaction to occur at room temperature. I do. After 5-20 minutes, the reaction is stopped with sulfuric acid.

(2) Measurement Measure the absorbance at 492 nm using an absorbance system for microplates. Ultimately, the more substances to be detected, the weaker the absorbance, so the substance is detected.

Problems to be Solved by the Invention As described in the previous section, conventional immunodetection methods require many steps, the reaction proceeds in a heterogeneous system in which a solid phase and a liquid phase coexist, and enzymes are used for the final detection. Because it uses a reaction,
In essence, it took a long time.

Means for Solving the Problems The antibody and the probe substance are combined by mixing an antibody that specifically binds to the target detection substance, a similar substance that binds to the antibody, and a rope substance possessed by the antibody, and the antibody and the probe substance are combined. quench the fluorescence. Thereafter, by introducing the target detection substance, the antibody and the probe substance are dissociated, and detection is performed using the phenomenon in which fluorescence increases again.

Effect By taking the above-mentioned means, the reaction proceeds in a homogeneous system (liquid phase), and furthermore, since no enzymatic reaction is used, it is possible to significantly shorten the detection time.

Examples Generally, antibodies emit fluorescence at around 340 nm upon excitation at 280 nm. This fluorescence is quenched by various quenching substances, but some of the most well-known quenching substances include nitrobenzene, dinitrobenzene, and trinitrobenzene. A similar effect can be obtained even if a functional group such as a carboxyl group, a carboxyl group, a hydroxyl group, or a thiol group is introduced. 2,4-dinitrofluorobenzene is readily available as a reagent for amino acid sequencing using the Sanger method, and is considered desirable because it easily binds to amino groups in both haptens and proteins.

Hereinafter, an example will be described which was carried out experimentally to confirm the principle of the present invention.

Example 1 First, a method for synthesizing a glove substance will be briefly described. Labor costs, drawings, single yen, Takahashi, Japanese Forensic Journal (Jpn, T
, , Legal Med, , 37(4), 41
7.1983), N-(4-aminobutyl)methamphetamine, (M
ANH2) was synthesized.

MANH2 and 2,4-dinitrofluorobenzene were mixed in equal cells in acetone, stirred for 1 hour, and then purified by preparative thin layer chromatography. As a result, MANH2DNP, a probe substance having the following structural formula, was obtained.

The antibody used in this example was an anti-methamphetamine monoclonal antibody (αMAMA) produced by the inventors.
B1). This antibody had an affinity for methamphetamine (MA) of approximately 1o. below,
We will describe the procedure for experimental detection.

■ Buffer (pH 7 phosphate buffer 0.45
aMAMABl was dissolved in a solution (filtered through a microfilter) to a concentration of 1x10M. Transfer 360 μL of this solution to a microcell for fluorescence measurement with a wavelength of 2sonm (bandpass 5nm).
) < 7) Excitation light emitted fluorescence with an intensity of approximately 4ts (FL○) at a fluorescence wavelength of 340nm (bandpass 1°nm) (first
Figure, part a).

■ MANH2DNP3x10 M(7)/<777
- Addition of 20 μL of solution causes quenching and decreases fluorescence intensity to 2
It decreased to 6 (FLl). This quenching reaction reached an equilibrium state in about 15 seconds (Fig. 1, part b).

■ When 20 μL of a 3×10 M buffer solution of MA (final concentration 1×10 M) was added to the above solution, the antibody and MA bound together and MANH2DNP was released, inhibiting quenching, resulting in a fluorescence intensity of approximately 40 μL (final concentration 1×10 M). FLx). This reaction reached equilibrium in about 30 seconds (Fig. 1, C
part).

As described above, MA was introduced into the solution at stage K and ■, and MA could be detected from the increase in fluorescence intensity. In order to confirm the detection sensitivity under these experimental conditions,
Figure 2 shows the changes in the translated light intensity when using the . In addition, in FIG. 2, the MA concentration is shown as the final concentration. The vertical axis is shown.

=6 From the results shown in Figure 2, it was proved that MA at a concentration of about 10 M could be detected by the method of the present invention.

Example 2 In place of MANH2DNP, a compound with the structural formula shown below was used as a probe substance, and the same operation as in Example 1 and -6,5 was carried out. Although the detection sensitivity fell to 1o, the same effect was obtained. It was done.

Example 3 As a result of carrying out the same operation as in Example 1 using a compound with the structural formula shown below as a glove substance instead of MANH2DNP, the detection sensitivity was lowered to 10-65 as in Example 2, but the same result was obtained. It worked.

The present invention has been explained above mainly using the detection of MA as an example, but it is of course a general method that can be applied to all other chemical substances. Moreover, although a derivative of dinitrobenzene was used as the fluorescence quenching substance for ease of implementation, it is easy to imagine that similar effects can be obtained with trinitrobenzene or nitrobenzene.

Effects of the Invention According to the present invention, it has become possible to shorten immunological detection, which conventionally required 5 hours, to less than 1 minute.

[Brief explanation of the drawing]

FIG. 1 is a graph showing temporal changes in the fluorescence intensity of an antibody when the present invention is carried out, and FIG. 2 is a graph showing changes in fluorescence quenching inhibition at each MA concentration. Name of agent: Patent attorney Toshio Nakao and one other person: So-and-so Aiko

Claims (4)

[Claims] (1) Using a probe substance in which an antibody that specifically binds to the target detection substance, a similar substance that binds to the antibody, and a fluorescence quenching substance that has the function of quenching the fluorescence of the antibody are chemically bonded, An immunodetection method that uses the phenomenon that the fluorescence of an antibody, which has been quenched by mixing the antibody and probe substance in advance, increases with the introduction of the target detection substance. (2) The immunodetection method according to claim 1, wherein the fluorescence quenching substance having the function of quenching fluorescence is nitrobenzene, dinitrobenzene, trinitrobenzene, or a derivative thereof. (3) The immunological detection method according to claim 1, wherein the target substance to be detected is methamphetamine, amphetamine, or ephedrine. (4) The immunodetection method according to claim 1, wherein the analogous substance that binds to the antibody has the following structural formula. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ However, n is 0 or a positive number of 1 or more, R1 is a hydrogen atom or a hydroxyl group, R2 is a hydrogen atom or an alkyl group,
X represents a hydrogen atom or a methoxy group.